Abstract: Pioneer transcription factors are master regulators of cell fate and direct the differentiation of the ~200 human cell types during development. For their role in controlling cellular fates, pioneer factors are used to artificially engineer cell states using cellular reprogramming technologies for induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) for biomedical and clinical applications. Advances in cryogenic electron microscopy (cryo-EM) could provide the first glimpses into how pioneer factors access nucleosome core particles. However, they are largely confined to SOX and POU pioneer factor families bound to peripheral parts of nucleosomes and the underpinnings for the mechanistic basis for other pioneer factors remain obscure. Here we elucidate the basis for the pioneer activity of the multi-domain pioneer factor KLF4—a C2H2 zinc finger protein that plays an indispensable role in stem cell pluripotency and differentiation. We performed quantitative binding assays of different KLF4 mutants to nucleosomes. We could uncover which domains of KLF4 are critical for nucleosome engagement and verify our findings in somatic cell reprogramming to induced pluripotent stem cells (iPSCs). Initial cryo-EM maps reveal that KLF4 exhibits an uncommon binding mode to nucleosomes. Unlike most pioneering factors that bind solely to the DNA, KLF4 binds both DNA and histone components. This suggests mechanism whereby KLF4 destabilizes packing of nucleosomes and triggers DNA unwrapping in a step-wise manner. Through mechanistic insights how pioneer factors resolve chromatin barriers, we design enhanced factors to better control cell fates and boost cellular programming and stem cell differentiation.
